This invention relates to compositions and methods for treating conditions responsive to estrogen. The compositions and methods utilize estrogen agonist/antagonist compounds. In both men and post-menopausal women, conditions such as rheumatoid arthritis, colon cancer, tissue wounds, skin wrinkles and cataracts are treated with the compositions and methods of the present invention.
In premenopausal women, 17xcex2-estradiol produced by the ovaries is the chief circulating estrogen. Serum estradiol concentrations are low in preadolescent girls and increase at menarche. In women, they range from about 100 pg per milliliter (367 pmol per liter) in the follicular phase to about 600 pg per milliliter (2200 pmol per liter) at the time of ovulation. They may rise to nearly 20,000 pg per milliliter (70,000 pmol per liter) during pregnancy. After menopause, serum estradiol concentrations fall to values similar to or lower than those in men of similar age (5 to 20 pg per milliliter [18 to 74 pmol per liter]) (Yen, S. S. C. and Jaffe, R. B., eds. Reproductive Endocrinology: Physiology, Pathophysiology and Clinical Management, 3rd ed. Philadelphia: W. B. Saunders, (1991)).
Steroidal estrogens are formed ultimately from either androstenedione or testosterone as immediate precursors. The reaction involves aromatization of the A ring, and it is catalyzed in three steps by a monooxygenase enzyme complex (aromatase) that uses NADPH and molecular oxygen as cosubstrates, (Miller, W. L., Endocr. Rev., 9:295-318 (1988)). In the first step of the reaction, C 19 (the angular methyl group residing on C 10 of the androgen precursor) is hydroxylated. A second hydroxylation results in the elimination of the newly formed C 19 hydroxymethyl group, and a final hydroxylation on C 2 results in the formation of an unstable intermediate that rearranges to form the phenolic A ring. The entire reaction consumes three molecules of NADPH.
Aromatase activity resides within a transmembrane glycoprotein (P450,arom) that is homologous with the cytochrome P450 family of monooxygenases (Nebert, D. W. and Gonzalez, F. J., Annu. Rev. Biochem. 56:945-993, (1987); Corbin, C. J., et al., Proc. Natl. Acad. Sci. USA, 85:8948-8952, (1988)); also essential is a ubiquitous flavoprotein, NADPH-cyctochrome P450 reductase. Both proteins are localized in the endoplasmic reticulum of ovarian granulosa cells, testicular Sertoli and Leydig cells, adipocytes, placental synctiotrophoblasts, the preimplantation blastocyst, and various brain regions, including the hypothalamus.
The ovaries are the principle source of estrogen in premenopausal women. The major secretory product is estradiol, synthesized by granulosa cells from androgenic precursors provided by thecal cells. Secreted estradiol is oxidized reversibly to estrone, and both of these estrogens can be converted to estriol. These transformations take place mainly in the liver, where interconversion between estrone and estradiol is catalyzed by 17-hydroxysteroid dehydrogenase.
In men and postmenopausal women, the principle source of estrogen is adipose tissue. In this and in other peripheral tissues, estrone is synthesized from dehydroepiandrosterone, which is secreted by the adrenal cortex. Thus, the contribution of adipose tissue estrogens is regulated, in part by the availability of androgenic precursors (Mendelson, C. R. and Simpson, E. R., Mol. Cell Endocrinol., 52:169-176, (1987)).
Autoimmune diseases, such as rheumatoid arthritis, involve aberrant regulation of cellular and humoral mediated immunity and are frequently associated with abnormal or enhanced T cell, B cell and macrophage effector functions directed towards self antigens. The activation of these cellular components towards self antigens is believed related to the break in feedback mechanisms associated with self tolerance. Autoimmune diseases encompass a whole spectrum of clinical entities and despite the differences in the target organ have many similarities. These include their preponderance in females of child bearing age with a female to male ratio varying from 50:1 in Hashimoto""s thyroiditis to 10:1 in systemic lupus erythematosus (SLE) to 2:1 in Myasthenia gravis (Ahmed et al., Am J. Path., 121:531 (1985)). In addition, these diseases are all characterized by the chronicity, the tendency of clinical remission and xe2x80x9cflare upsxe2x80x9d for poorly understood reasons, and the involvement of other organs. While the presence of autoantibodies, inappropriate expression of class II antigens, macrophage activation and T cell infiltration to the target organ have been described in essentially all of the autoimmune diseases, neither the triggering mechanisms which result in disease activation nort disease progression are well understood. Accordingly, therapy for these diseases is largely unsatisfactory and involves the use of gold salts, methotrexate, antimalarials, glucocorticoids (methylprednisolone), and other immunosuppressives as well as plasmaphoresis and attempts at inducing tolerance. Treatment of autoimmune diseases has not improved significantly over the past decade and primarily is associated with the use of nonsteroidal and steroidal anti-inflammatory agents to treat the symptoms of the disease. Clearly while suppression of the specific immune response directed against the host is necessary, generalized immunosuppression as with glucocorticoids has major liabilities in terms of side effect profile and the propensity of the immunosuppressed subject to be at greater risk for other infectious and non-infectious diseases.
Polymorphonuclear leukocytes (PMNL) play a regulatory role in inflammatory diseases. These cells, when activated, synthesize and release oxygen-centered molecules, chemo-attractants, and hydrolytic enzymes. There is evidence that the oxygen-centered molecules play a detrimental role in a number of diseases such as chronic inflammatory diseases, rheumatoid arthritis, SLE, and others. In the case of an autoimmune disease, SLE, for example, the initiation of an inflammatory response is self antigen stimulating one""s host neutrophils or PMNLs to secrete strong oxidants which damage surrounding cells and tissue.
Estrogen appears to be involved with autoimmune diseases although its role in disease progression or regression is complex and dependent on the nature of the autoimmune disease. Estrogen for example appears to have ameliorating effect on rheumatoid arthritis while having an exacerbating effect on systemic lupus (Chander and Spector; Ann. Rheum. Dis. 50:139). As reported by Jansson (Free Rad. Res. Comms., 14(3):195-208, (1991)), estrogen increased the activity of an enzyme generated by PMNLS, myeloperoxidase, which regulates the production of oxidants from hydrogen peroxide. This enzyme converts hydrogen peroxide to hypochlorous acid, a strong oxidant. By increasing the enzyme""s activity, and thus the presence of hypochlorous acid, the likelihood of increased oxidative stress on tissues, cells and various macromolecules in chronic inflammatory/autoimmune diseases is enhanced.
EP 664 125 A1 reports that inhibition of myeloperoxidase may be accomplished by treatment with certain 3-aroyl benzothiophines. Excess myeloperoxidase is associated with conditions which include systemic lupus erythematosis, Hashimoto""s thyroiditis, myasthenia gravis, rheumatoid arthritis and multiple sclerosis.
Estrogen has been demonstrated to have a suppressive role on T cell function and yet an immunostimulatory effect on B cells. Therefore, estrogen-like compounds should prove beneficial in diseases associated with activated T cells including rheumatoid arthritis, multiple sclerosis, Guillan Barre syndrome and Hashimoto""s thyroiditis through inhibition of T cell function (Holmadahl, J., Autoimmun. 2:651 (1989).
In addition to the suppressive effects of estrogen on T cells, estrogen may have additional protective roles. Marui et al., (J. Clin. Invest. 92:1866 (1993)) have recently reported that antioxidants suppress endothelial expression of VCAM-1. VCAM-1 is the ligand for VLA4, the T cell and macrophage integrin associated with trafficking of these cells out of the vasculature and into the perivascular space and target organs. As estrogen is an antioxidant, it would be anticipated that estrogen and related analogs will inhibit VLA-4 dependent trafficking of cells and thus hinder the immune cascade associated with autoimmune mediated disease.
Estrogen plays a detrimental role in other autoimmune diseases including systemic lupus and glomerulonephritis, diseases associated with immune complexes. While the mechanism(s) responsible for estrogen mediated disease progression are not known, the ability of estrogen to increase Fc mediated phagocytosis (Friedman et al., J. Clin. Invest. 76:162 (1985), and class 11 antigen expression and IL-1 production by macrophages from estrogen treated rodents (Flynn, Life Sci., 38:2455 (1986) has been reported. Enhancement of these macrophage mediated effector functions would be expected to contribute towards the immune cascade associated with self destruction.
Cancer of the large bowel is second only to lung cancer as a cause of cancer death in the United States. Approximately 133,500 new cases occurred in 1996, resulting in 54,900 deaths. The incidence rate for this extremely common malignant condition has not changed substantially during the past 40 years, although, for some reason, the mortality rate has decreased in recent years, particularly in females. Colorectal cancer generally occurs in individuals 50 years of age or older.
Most colorectal cancers, regardless of etiology, are believed to arise from adenomatous polyps. A polyp is a grossly visible protrusion from the mucosal surface and may be classified pathologically as a nonneoplastic hamartoma (juvenile polyp), a hyperplastic mucosal proliferation (hyperplastic polyp), or an adenomatous polyp. Only adenomas are clearly premalignant, and only a minority of such lesions ever develop into cancer. Population-screening studies and autopsy surveys have revealed that adenomatous polyps may be found in the colons of about 30 percent of middle-aged or elderly people. Based on this prevalence and the known incidence of colorectal cancers, it appears that fewer than 1 percent of polyps ever become malignant. Most polyps produce no symptoms and remain clinically undetected. Occult blood in the stool may be found in fewer than 5 percent of subjects with such lesions.
A number of molecular changes have been described in DNA obtained from adenomatous polyps, dysplastic lesions, and polyps containing microscopic foci of tumor cells (carcinoma in situ), which are thought to represent a multistep process in the evolution of normal colonic mucosa to life-threatening invasive carcinoma. These developmental steps towards carcinogenesis include point mutations in the K-ras proto-oncogene; hypomethylation of DNA, leading to gene activation; loss of DNA (xe2x80x9callelic lossxe2x80x9d) at the site of a tumor suppressor gene [the adenomatous polyposis coli (APC) gene] located on the long arm of chromosome 5 (5q21); allelic loss at the site of a tumor suppressor gene located on chromosome 18q [so-called the deleted in colorectal cancer (DCC) gene]; and allelic loss at chromosome 17p, associated with mutations in the p53 tumor suppressor gene. Thus, the altered proliferative pattern of the colonic mucosa, which results in progression to a polyp and then to carcinoma, may involve the mutational activation of an oncogene followed by and coupled with a loss of genes that normally suppress tumorigenesis. While the present model includes five such molecular alterations, others are likely involved in the carcinogenic process. It remains uncertain whether the genetic aberrations always occur in a defined order. Based on this model, however, it is believed that neoplasia develops only in those polyps in which all of these mutational events take place. (Mayer, R. J., Gastrointestinal Tract Cancer, Chapter 92, in Harrison""s Principles of Internal Medicine, 14th ed., 1998).
Several orally administered synthetic and naturally occurring materials have been assessed as possible inhibitors of colon cancer. The most effective class of these chemopreventive agents is aspirin and other nonsteroidal anti-inflammatory drugs, which are thought to suppress cell proliferation by inhibiting prostaglandin synthesis. Case-control studies have indicated that regular aspirin use reduces the risk for colonic adenomas and carcinomas as well as for death from large-bowel cancer; this inhibiting effect on colonic carcinogenesis appears to increase with the duration of drug use. While antioxidant vitamins such as ascorbic acid, tocopherols, and xcex2-carotene are present in diets rich in fruits and vegetables, which have been associated with lower rates of colorectal cancer, they have been found to be ineffective in a prospectively randomized trial as a means of reducing the incidence of subsequent adenomas in subjects who had undergone the removal of a colonic adenoma. Estrogen replacement therapy has been associated in prospective cohort studies with a reduction in the risk of colorectal cancer in women, conceivably by an effect on bile acid synthesis and composition. The otherwise unexplained reduction in colorectal cancer mortality in women may be a result of the widespread use of estrogen replacement in postmenopausal individuals. (Mayer, R. J., Gastrointestinal Tract Cancer, Chapter 92, in Harrison""s Princilles of Internal Medicine, 14th ed., 1998).
Wound healing is usually a coordinated, stereotyped sequence of events that includes (a) tissue disruption and loss of normal tissue architecture; (b) cell necrosis and hemorrhage; hemostasis (clot formation); (c) infiltration of segmented and mononuclear inflammatory cells, with vascular congestion and tissue edema; (d) dissolution of the clot as well as damaged cells and tissues by mononuclear cells (macrophages) (e) formation of granulation tissue (fibroplasia and angiogenesis). This sequence of cellular events has been observed in wounds from all tissues and organs generated in a large number of mammalian species (Gailet et al., 1994, Curr. Opin. Cell. Biol. 6:717-725).
Estrogen accelerates endothelial cell growth in vitro and in vivo (Morales, D. E., et al., Circulation, 91:755-63 (1995); Krasinski, K., et al., Circulation, 95:1768-72 (1997)). The rapid reendothelialization induced by estrogen after vascular injury may be due in part to increased local expression of vascular endothelial growth factor. Estrogen also inhibits apoptosis of cultured human endothelial cells in an estrogen receptor-dependent manner (Spyridopoulos, I., et al., Circulation, 95:1505-14 (1997)). Early restoration of endothelial integrity by estrogen may contribute to the attenuation of the response to injury by increasing the availability of nitric oxide, which can directly inhibit the proliferation of smooth-muscle cells (Cornwell, T. L., et al., Am. J. Physiol., 267:C1405-C1413 (1994)). Estrogen directly inhibits the migration and proliferation of smooth-muscle cells in vitro (Kolodgic, F. D., et al., Am. J. Pathol. 148: 969-76 (1996); Bhalla, R. C., et al., Am. J. Physiol., 272:H1996-H2003 (1997)).
Currently available wound healing therapies involve the administration of therapeutic proteins. Such therapeutic proteins may include regulatory factors involved in the normal healing process such as systemic hormones, cytokines, growth factors and other proteins that regulate proliferation and differentiation of cells. Growth factors, cytokines and hormones reported to have such wound healing capacity include, for example, the transforming growth factor-xcex2 superfamily (TGF-xcex2) of proteins (Cox, D. A., Cell Biology International, 19:357-371 (1995)) acidic fibroblast growth factor (FGF) (Slavin, J., Cell Biology International, 19:431-444 (1995)), macrophage-colony stimulating factor (M-CSF) and calcium regulatory agents such as parathyroid hormone (PTH).
Epidemological evidence suggests that estrogens may protect against cataracts. Although women are at higher risk of developing cataracts than are men, this increased risk comes after menopause, when estrogen has waned Livingston, P. M., et al., Dev. Opthalmol. 26:1-6, (1994); Klein, B. E., et al., Arch. Ohthalmol. 116:219-225, (1998)). In one study of 544 women, early onset of menopause was associated with a 2.9-fold risk of developing cataracts (Shibata, T., et al., Dev. Opthalmol. 26:25-33, (1994)). Moreover, the results of three small epidemiological studies suggest that postmenopausal estrogen replacement therapy reduces the incidence of cataracts (Klein, B. E., et al., Arch. Ohthalmol. 112:85-91, (1994); Cumming, R. G. and Mitchell, P., Am. J. Epidemiol., 145:242-249, (1997); Benitez del Castillo, J. M., et al., Ophthalmology, 104:970-973, (1997)). An in vivo rat model of age-related cataracts suggests that the protective effect of estrogen is a genomic one (Bigsby, R. M., Proc. Natl. Acad. Sci. USA, 96:9328-9332, (1999)).
Breast cancer is a hormone-dependent disease. Women without functioning ovaries who never receive estrogen replacement do not develop breast cancer. The female-to-male ratio for the disease is about 150 to 1. A host of findings indicate that hormones play a critical role as promoters of the disease. For most epithelial malignancies, a logxe2x80x94log plot of incidence versus age shows a straight-line increase with every year of life. A similar plot for breast cancer shows the same straight line increase, but with a decrease in slope beginning at the age of menopause. The three dates in a woman""s life that have a major impact on breast cancer incidence are age of menarche, age at first full-term pregnancy, and age of menopause. Women who experience menarche at age 16 have only 50 to 60 percent of the lifetime breast cancer risk of women who experience menarche at age 12. Similarly, menopause occurring 10 years before the median age (52 years), whether natural or surgically induced, reduces lifetime breast cancer risk by about 35 percent. Compared with nulliparous women, women who have a first full-term pregnancy by age 18 have 30 to 40 percent the risk of breast cancer. Thus, length of menstrual lifexe2x80x94particularly the fraction occurring before the first full-term pregnancyxe2x80x94is a substantial component of the total risk of breast cancer. This factor can account for 70 to 80 percent of the variation in breast cancer frequency in different countries.
International variation has provided some of the most important clues on hormonal carcinogenesis. A woman living to age 80 in North America has 1 chance in 9 of developing invasive breast cancer. Asian women have one-fifth to one-tenth the risk of breast cancer of women in North America or Western Europe. Asian women have substantially lower concentrations of estrogens and progesterone. These differences cannot be explained on a genetic basis, because Asian women living in a Western environment have a risk identical to that of their Western counterparts. These women also differ markedly in height and weight from Asian women in Asia; height and weight are critical regulators of age of menarche and have substantial effects on plasma concentrations of estrogens. (Lippman, M. E., Breast Cancer, Chapter 91, in Harrison""s Principles of Internal Medicine, 14th ed., 1998).
Menopause occurs naturally at an average age of 50 to 51 years in the USA. As ovaries age, response to pituitary gonadotropins (follicle-stimulating hormone [FSH] and luteinizing hormone [LH]) decreases, initially resulting in shorter follicular phases (thus, shorter menstrual cycles), fewer ovulations, decreased progesterone production, and more irregularity in cycles. Eventually, the follicle fails to respond and does not produce estrogen. The transitional phase, during which a woman passes out of the reproductive stage, begins before menopause. It is termed the climacteric or perimenopause, although many persons refer to it as menopause.
Premature menopause refers to ovarian failure of unknown cause that occurs before age 40. It may be associated with smoking, living at high altitude, or poor nutritional status. Artificial menopause may result from oophorectomy, chemotherapy, radiation of the pelvis, or any process that impairs ovarian blood supply.
Symptoms of the climacteric range from nonexistent to severe. Hot flushes (flashes) and sweating secondary to vasomotor instability affect 75% of women. Most have hot flushes for more than 1 year, and 25 to 50% for more than 5 years. The woman feels warm or hot and may perspire, sometimes profusely. The skin, especially of the head and neck, becomes red and warm. The flush, which may last from 30 sec to 5 min, may be followed by chills. Vasomotor symptoms of the hot flush coincide with the onset of LH pulses, but not every increase in LH is associated with a hot flush, suggesting that hypothalamic control of LH pulses is independent of that of flushes. This independence is confirmed by the occurrence of hot flushes in women who have had pituitary failure and do not secrete LH and/or FSH.
Psychologic and emotional symptomsxe2x80x94including fatigue, irritability, insomnia, inability to concentrate, depression, memory loss, headache, anxiety, and nervousness and timidity can occur. Sleep disruption by recurrent hot flushes contributes to fatigue and irritability. Intermittent dizziness, paresthesias, palpitations, and tachycardia may also occur. Nausea, constipation, diarrhea, arthralgia, myalgia, cold hands and feet, and weight gain are also common.
The large reduction in estrogen leads to profound changes in the lower genital tract; e.g., the vaginal mucosa and vulvar skin become thinner, the normal bacterial flora changes, and the labia minora, clitoris, uterus, and ovaries decrease in size. Inflammation of the vaginal mucosa (atrophic vaginitis) can cause the mucosa to have a strawberry appearance and can lead to urinary frequency and urgency, vaginal dryness, and dyspareunia. Women tend to lose pelvic muscle tone and to develop urinary incontinence, cystitis, and vaginitis.